Molecularly imprinted polymers selective for tobacco specific nitrosamines and methods of using same

ABSTRACT

A class of molecularly imprinted polymers that specifically recognizes and binds to TSNAs are useful, for example, in analysis and separation of TSNAs from biological fluids. Such polymers are also useful in methods of treating and manufacturing tobacco products and materials.

This application is a continuation of U.S. patent application Ser. No.14/175,885, filed Feb. 7, 2014, now issued as U.S. Pat. No. 8,889,795,which is a continuation of U.S. patent application Ser. No. 12/518,051,filed Nov. 5, 2010, now issued as U.S. Pat. No. 8,733,369, which is theNational Stage of International Application No. PCT/EP2007/062781,titled “Molecularly Imprinted Polymers Selective for Tobacco SpecificNitrosamines and Methods of Using the Same,” filed Nov. 26, 2007, whichin turn claims priority to Swedish Patent Application NumberSE0602625-6, filed Dec. 7, 2006. The entire contents of theaforementioned applications are herein expressly incorporated byreference.

FIELD OF THE INVENTION

Generally, the present invention relates to molecularly imprintedpolymers and use of the polymers in bioanalysis and separation ofnicotine metabolites. More specifically, the invention relates tomolecularly imprinted polymers having specificity for tobacco specificnitrosamines and includes methods of using the polymers to treattobacco, tobacco substitutes, and their derivatives to reduce the levelof targeted compounds therein.

BACKGROUND OF THE INVENTION

In the fields of medical, dietary, environmental and chemical sciencesthere is an increasing need for the selective separation of specificsubstances from complex mixtures of related substances. The aim can bethe quantitative extraction of a certain compound or compounds, themeasurement of their concentration or the selective removal of a targetcompound from a multi-component mixture.

Stricter health controls have increased the demand for methods allowingsensitive and selective quantification of hazardous products andmetabolites from certain everyday substances in widespread use. Ofparticular concern are chemical compounds related to use oftobacco-based products, which compounds are either originally present inthe raw tobacco leaf itself or generated during the smoking process.Nitroso-containing compounds, such as nitrosamines, are regarded asbeing of special significance in this regard.

With the aim of reducing the occurrence of hazards related to smoking,certain pharmaceutical products have been produced containing only theneuroactive substance, nicotine, the chemical claimed to be responsiblefor the dependence aspects of smokable material.

Among the nicotine formulations for smoking cessation therapy, nicotinechewing gum has found the most widespread use. The quality controlrequired during production includes monitoring of nicotine levels aswell as monitoring of the primary nicotine oxidation products cotinine,myosmine, nicotine-cis-N-oxide, nicotine-trans-N-oxide andbeta-nicotyrine. Quantitation of nornicotine, anatabine and anabasine isalso desirable, if not required. Improved methods and materials for suchmonitoring and quantitation are needed in the art. Use of such cigarettesubstitutes can cause nitrosamine nicotine metabolites to be produced invivo by natural metabolic processes during the residence of the nicotinewithin body tissues. The levels of these metabolites remain below theconcentrations at which most analytical procedures can performquantitatively. So in addition to methods and materials for use duringproduct manufacture, there remains a need for improved ways to monitorthe low levels of nicotine metabolites in vivo.

Along with the needs felt in relation to newer products, traditionaltobacco products also require methods and materials for quantifying,reducing or removing components from tobacco or tobacco smoke. Suchcomponents include tobacco specific nitrosamines (TSNAs) and theiralkaloid precursors: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(“NNK”), 4-(methylnitrosamino)-4-(3-pyridyl)butanal (“NNA”),N-nitrosonornicotine (“NNN”), N-nitrosoanabasine (“NAB”),N-nitrosoanatabine (“NAT”),4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (“NNAL”),4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (“iso-NNAL”), and4-(methylnitrosamino)-4-(3-pyridyl)butanoic acid (“iso-NNAC”).

To properly quantify the amount of such compounds present in humanbiological fluids, methods are being developed to analyse the alkaloids,especially the nitrosylated decomposition products and metabolites intobacco. Existing chromatographic separation or extraction methods usedfor this analysis lack the robustness, sensitivity and speed required inorder to handle the large number of samples generated when screening thegeneral population. With existing methods, the low concentration ofnitrosamines, typically present in picograms per milliliter, demandsextensive sample preparation with multi-step extractions and oftenchemical derivatization (for example deuteration prior to massspectrometry) of the analyte prior to analysis. One reason for thiscomplexity is that existing separation materials are not selective as,for example, an antibody or biological receptor might be for themetabolites in question but rather rely on physico-chemical propertieslike charge or hydrophobicity of the metabolites for the separationbehaviour. These physico-chemical properties may be shared by many otherirrelevant molecules in the sample. A quick and simple method for theanalysis of TSNAs is therefore a significant unmet medical analyticalneed.

During recent years numerous reports of selective recognition of smallmolecules with materials prepared by molecular imprinting (molecularlyimprinted polymers or MIPs) have appeared. MIPs are polymers havingreactive sites adapted to bind selectively with targeted compounds.Non-covalently prepared molecularly imprinted materials have been usedfor chiral recognition of a variety of small molecules includingtherapeutic drugs, sugars, nucleotide bases, and pesticides as well assteroid and peptide hormones. The high affinity and selectivity for thetarget analyte exhibited by some of the imprinted materials havejustified a comparison with the corresponding immuno-affinity (IA)phases. In contrast to the latter phases however, the MIP materials arestraightforward to prepare, stable in most media and reusable over longperiods of time. Applications of the MIP materials in chromatography,separation (continuous or batch), chemical sensing or in specific assaysare therefore under investigation.

Another application is solid-phase extraction (SPE) of analytes presentin low concentrations in biological samples, or in complex matrices. SPEmay lead to selective enrichment and clean-up of an analyte to levelsnot achievable with existing methods. Molecularly imprinted solid phaseextractions (MISPE) have been used in bioanalysis, food analysis andenvironmental analysis. In these examples selective enrichment andclean-up of the analyte is obtained resulting in higher accuracy and alowering of the detection limit (LOD) in the subsequent chromatographic(eg HPLC) or mass spectrometric quantification.

In view of their high selectivity combined with good affinity for thetarget molecule or a group of target molecules, MIPs have attractedconsiderable interest from the food industry as a tool to improve foodquality. This requires the use of a MIP for selective removal ofundesirable components from the food matrix. Since these components areoften present in low concentrations, the saturation capacity of the MIPis typically not a limiting factor.

According to WO 05/112670, which is specifically incorporated byreference herein, it can be preferred to have MIP material capable ofselectively absorbing the most common nitrosylated nicotine derivativesfrom complex matrices such as urine, giving quantitative recovery andthereby leading to low errors in the estimation of chemicalconcentrations. The examples of WO 05/112670 are limited to MIPsprepared using acidic or highly acidic template monomers such asmethacrylic acid (MAA), trifluoromethacrylic acid (TFMAA),4-vinylbenzoic acid, and 4-vinyl benzene sulphonic acid.

In addition to quantification it is also well known to attempt to reducethe harmful effects of consuming material containing tobacco, tobaccosubstitutes or mixtures thereof by reducing the levels of targetedcompounds. Such reductions can be made in the material itself or in aderivative thereof such as an extract of the material. Reduction canalso be effected in the thermal decomposition products of the material,i.e. mainstream and sidestream smoke obtained by combustion, or theaerosols produced by heating the material to a temperature below itscombustion temperature.One well known method for this sort of reduction is to contact thethermal decomposition products of the material with a filter thatadsorbs undesired components therefrom. An alternative method involvessolvent extraction of the material, for example as disclosed in the USpatent specification U.S. Pat. No. 5,601,097. According to thatspecification, the protein content of tobacco material is reduced bytreating the tobacco with a solution containing a surfactant to extractpolypeptides, separating the solution, removing the surfactant and thepolypeptides from the solution, and recombining the solution with thetobacco material. International patent specification WO 01/65954discloses a process in which tobacco is contacted with a supercriticalextraction fluid such as supercritical carbon dioxide to selectivelyreduce or eliminate nitrosamines.

These processes are equally applicable to both tobacco itself and totobacco substitutes, i.e., natural or synthetic materials having similarcharacteristics to natural tobacco that enable them to be consumed in asimilar way to tobacco, whether by smoking, chewing, inhaling orotherwise.

There has been an attempt to remove nicotine from tobacco smoke usingMIPs, as reported in Liu, Y., et al., Molecularly imprinted Solid-PhaseExtraction Sorbent for Removal of Nicotine from Tobacco Smoke,Analytical Letters, Vol. 36, No. 8, pp 1631-1645 (2003). The MIPdescribed in the article was designed to bind nicotine and not the moretoxic nicotine metabolites such as nitrosamines. It is unclear if theMIP was in fact selective for nicotine as the scientific methodproducing the data was lacking in key control-checking elements. Asdescribed in WO 05/112670, MIPs selective for TSNAs can be used to treattobacco products and thereby reduce the levels of one or morenitroso-containing compounds from the tobacco product. Such MIPs furtherfind use in the analysis and quantification of TSNAs in vivo, commonlyin relation to consumption of tobacco products, and in the preparationand evaluation of non-tobacco products. So despite advances thereremains a need in the art for novel MIPs and methods of employing thesame in the field of nicotine and nicotine metabolites.

SUMMARY OF THE INVENTION

The present invention meets the needs in the art by providing uniqueMIPs which are particularly selective for nitroso-containing compounds.

MIPs of the invention can be obtained by co-polymerising a neutralfunctional monomer or monomers and a hydrophobic cross-linker in thepresence of a structural analogue of a nitrosamine, in a polymerizationmedium containing a free radical initiator, after which the template isremoved from the MIP.

The invention includes the use of the MIPs for analytical andpreparative extractions, in chromatography, for analytical samplepre-treatment, in chemical sensors or as a solid phase filter forextraction of TSNAs from nicotine-containing substances or devices.

According to one embodiment, a molecularly imprinted polymer selectivefor at least one tobacco specific nitrosamine (TSNA) is provided, thepolymer having been prepared using materials comprising a TSNA or astructural analogue thereof, a neutral functional monomer, and ahydrophobic cross-linker. The structural analogue of a TSNA could be anenamine analogue of a TSNA or a sulfonamide analogue of a TSNA or anamide analogue of a TSNA, e.g. a formamide analogue of a TSNA. Theneutral functional monomer could be selected from the group consistingof 2-hydroxyethylmethacrylate (HEMA), acrylamide, methacrylamide,glycerol monoacrylate, and glycerol monomethacrylate. The hydrophobiccross-linker could be selected from the group consisting of ethyleneglycol dimethacrylate (EDMA), trimethylolpropane trimethacrylate (TRIM),and divinylbenzene (DVB).

In the embodiment of the invention, the polymer could be selective forNNK, NNA, NNN, NAB, NAT, NNAL, iso-NNAL, or iso-NNAC.

According to a further embodiment of the invention, a smoking article isprovided, comprising a smoking material and a molecularly imprintedpolymer according to the embodiment described above.

According to a further embodiment of the invention, a smoke filter cancomprise a molecularly imprinted polymer according to the above.

According to a further embodiment of the invention, a kit can comprise amolecularly imprinted polymer according to the above and instructionsfor using the molecularly imprinted polymer to perform at least one ofdetecting, quantifying, and separating nitrosamines in a sample.

According to a further embodiment of the invention, a method ofpreparing a molecularly imprinted polymer selective for TSNAs isprovided, comprising co-polymerizing at least one neutral functionalmonomer and at least one hydrophobic cross-linker in the presence of atleast one TSNA structural analogue in a polymerization medium containingat least one free radical initiator to produce a molecularly imprintedpolymer bound to a TSNA structural analogue and removing the TSNAstructural analogue from the molecularly imprinted polymer.

According to a further embodiment of the invention, a method of reducingthe level of at least one TSNA in a tobacco product is provided,comprising treating the tobacco product with a molecularly imprintedpolymer according to the invention. The tobacco product could beproduced by the thermal decomposition of a material containing tobacco,a tobacco substitute or a mixture thereof, for example, by heating thematerial to a temperature below its combustion temperature, bycombustion of the material, or by contacting a material containingtobacco, a tobacco substitute or a mixture thereof with a solvent.

According to a further embodiment of the invention a method ofmanufacturing a tobacco material is provided, comprising the steps oftreating a material containing tobacco, tobacco substitute or a mixturethereof with a solvent to produce an extract, contacting the extractwith a molecularly imprinted polymer according to the invention so as toreduce the level thereof in the extract, and combining the treatedextract with the extracted tobacco material.

In this specification, “tobacco product” means a material containingtobacco (including tobacco leaf or tobacco stem), or a tobaccosubstitute, or a blend of tobacco and tobacco substitutes, andderivatives of such material, including extracts of the material, smokeproduced by thermal decomposition of the material and aerosols producedby heating the material to below its combustion temperature.

Where the tobacco product is a derivative produced by the thermaldecomposition of material containing tobacco or a tobacco substitute,the decomposition may be effected by combustion of the material, as in aconventional cigarette, or by heating the material to a temperaturebelow its combustion temperature, in accordance with a process used insome known alternative tobacco products in order to produce an aerosolthat is inhaled by the consumer.

Alternatively, the tobacco product may be a derivative produced bycontacting material containing tobacco or a tobacco substitute with asolvent. In particular, the invention provides a method of manufacturinga material for smoking comprising the steps of extracting smokablematerial with a solvent, treating the extract with a MIP selective forat least one nitroso-compound to reduce the level thereof in the extractand combining the treated extract with the smokable material.

In this process, the smokable material may be in any convenient form,for example fines, stems, scraps, cut lamina, shredded stems, or anycombination thereof. The solvent may be aqueous or non-aqueous, such asmethanol, ethanol or a super-critical fluid extraction medium, such assuper-critical carbon dioxide liquid. The extraction may be carried outunder any conditions favoring the extraction of nitrogen-containingcompounds from tobacco.

The invention also includes a smoking article comprising tobacco ortobacco substitute and a MIP selective for the removal of at least onenitroso-containing compound from the thermal decomposition productthereof.

The smoking article of the invention may take any conventional form, forexample a cigarette, cigar or cigarillo. In particular the smokingarticle may comprise a rod of smoking material optionally in a wrapper,with or without a filter. The wrapper may be of paper, tobacco leaf,reconstituted tobacco or a tobacco substitute. Alternatively, where, forexample, the smoking article is intended to produce low emissions ofsidestream smoke, or lower levels of pyrolysis products in themainstream smoke, the wrapper may be composed of non-combustibleinorganic material such as a ceramic material. The filter may be of anysuitable material, for example fibrous cellulose acetate, polypropyleneor polyethylene, or paper.

The smoking material is preferably tobacco but may be a tobaccosubstitute such as non-tobacco smoking material. Examples of non-tobaccosmoking materials are dried and cured vegetable material, includingfruit materials, and a synthetic smoking material such as may beproduced from alginates and an aerosol-generating substance such asglycerol. The smoking material may also comprise a blend of tobacco andnon-tobacco smoking materials. Where the smoking material comprisestobacco, the tobacco may be of any suitable type, or a blend thereof,including air-cured, fire-cured, flue-cured, or sun-cured lamina orstem, and may have been processed using any appropriate process. Forexample, the tobacco may be cut, shredded, expanded or reconstituted.The smoking material may also include conventional additives, such asameliorants, colorants, humectants (such as glycerol and propyleneglycol), inert fillers (such as chalk), and flavourings (such as sugar,liquorice and cocoa).

The invention may also be applied to tobacco that is intended for oralor nasal consumption by sucking, chewing, or nasal ingestion, ratherthan smoking. Such products include snuff, snus, and chewing tobacco.

The MIP may be incorporated in the smokable material. Accordingly, theinvention includes smoking material containing a MIP selective for theremoval of at least one tobacco specific nitrosamine from the thermaldecomposition products of the smokable material. Alternatively, wherethe smoking article comprises a rod of smokable material in a wrapper,the MIP may be incorporated in the wrapper. The invention thereforeincludes wrapper material for smoking articles comprising amolecularly-imprinted polymer selective for the removal of a targetedcomponent from the thermal decomposition products of a smoking material.The wrapper may be a cellulose-based material such as a paper or atobacco based material such as reconstituted tobacco.

The preferred smoking articles of the invention are cigarettes,comprising a rod of tobacco, wrapper, and a filter including a MIPselective for the removal of at least one tobacco specific nitrosaminefrom the thermal decomposition products of a smokable material.

The invention also includes a smoke filter comprising a MIP selectivefor the removal of at least one tobacco specific nitrosamine from thethermal decomposition products of a smoking material. The smoke filtermay be produced separately from the smoking article, for example in theform of a cigarette or cigar holder, or it may be integrated into thesmoking article, for example in the form of a cigarette with a filtertip.

Smoke filters in the form of filter tips may be of any conventionalconstruction. For example a “dalmatian” type filter comprising a sectionof fibrous filter material, such as cellulose acetate, the MIP being inparticulate form and distributed throughout the section. Alternativelythe filter may be in the form of a “cavity” type filter, comprisingmultiple sections wherein the MIP may lie between two adjacent sectionsof fibrous filter material. The smoke filter may also comprise otheradsorbent materials such as an ion-exchange resin, a zeolite, silica,alumina or amberlite.

In use, the smoke passes through the filter, the MIP selectively adsorbsand retains the targeted compounds from the smoke and the filtered smokeis delivered to the smoker.

The smoke filters and smoking articles according to the invention mayinclude means for protecting the MIP from, or reducing its exposure to,smoke when in use. This may be achieved in a number of different ways.For example the smoke filter may comprise a filter element for adsorbingmaterials from the vapour or particulate phase of smoke. Such filterelements may comprise a general adsorbent such as activated carbon,which may be in any convenient form, such as threads, particles,granules, cloth, or paper. The filter element may also be a selectiveadsorbant such as an ion-exchange resin, a zeolite, silica, alumina oramerlite. The means for protecting the MIP may include two or more suchfilter elements of different compositions, for example a first filterelement of cellulose acetate, and a second filter element of activatedcarbon. The provision of multiple filter elements in smoke filters andsmoking articles is well known, and any conventional configuration offilter, and associated methods of construction, may be used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an outline of the procedure for synthesis of an imprintedpolymer;

FIG. 2 shows tobacco specific nitrosamines;

FIG. 3 shows isosteric analogues of nitrosamines;

FIG. 4A shows examples of amide and sulfonamide based target analogs;

FIG. 4B shows an enamine target analogue (MPAPB) used as a template toprepare a MIP for extraction of NNAL;

FIG. 5 shows the chemical structure of 2-hydroxyethyl methacrylate;

FIG. 6 shows the chemical structure of ethylene glycol dimethacrylate;

FIG. 7 shows results from extraction of N-nitrosopiperidine and nicotinewhere template 1 is formamide, template 2 is enamine and template 3 issulfonamide;

FIG. 8 shows percentage of TSNAs and nicotine extracted from aqueoussolution;

FIG. 9 shows percentage of TSNAs released upon a water wash;

FIG. 10 shows percentage of TSNAs and nicotine extracted from aqueoussolution;

FIG. 11 shows percentage of TSNAs released upon a water wash;

FIG. 12 shows the percentage of nicotine unretained after five separatesample loads;

FIG. 13 shows the percentage of NNN unretained after five separatesample loads;

FIG. 14 shows the percentage of NNK unretained after five separatesample loads;

FIG. 15 shows the percentage of NAT unretained after five separatesample loads;

FIG. 16 shows the percentage of NAB unretained after five separatesample loads;

FIG. 17 is a side elevation, partly longitudinal cross-section andpartially broken away view of a smoking article with a smoke filteraccording to the invention; and

FIG. 18 is a similar view to FIG. 17 of a smoking article with analternative smoke filter according to the invention.

In the drawings, similar features are given like reference numerals.

DETAILED DESCRIPTION OF THE INVENTION

Molecular imprinting typically consists of the following steps: (1) atemplate compound, which may be the targeted molecule or a structuralanalogue thereof, is allowed to interact with a selected functionalmonomer, or monomers, in solution to form a template-monomer complex;(2) the template-monomer complex is co-polymerized with a cross-linkingmonomer resulting in a polymeric matrix incorporating the templatecompound; (3) the template compound is extracted from the polymer matrixto form a MIP that can be used for selective binding of the targetedmolecule.

Prior to step (3), where the MIP is prepared as a solid polymer (ormonolith) it is typically crushed and sieved to obtain a desired sizefraction of particulate material. When prepared by either suspension oremulsion polymerization methods, such crushing and sieving isunnecessary since the particle size can be controlled within the desiredlimits during the polymerization process. Particulate material preparedby any of the aforementioned methods can be packed into achromatographic or solid phase extraction column and used forchromatographic separation of the template from other components of amixture, including molecules with similar structures or functionalities.

The reactive sites on the molecularly imprinted polymer exposed byremoval of the template compound will be in a stereo-chemicalconfiguration appropriate for reaction with fresh molecules of thetargeted molecule. As a result, the molecularly imprinted polymer can beused for selective binding of the targeted molecule.

The ‘non-covalent’ route has been widely used to generate molecularlyimprinted binding sites. This makes use of non-covalent self-assembly ofthe template compound and functional monomers to form thetemplate-monomer complex, followed by free radical polymerization in thepresence of a cross-linking monomer and finally template compoundextraction. Covalent imprinting, in which the template molecule and asuitable monomer or monomers are covalently bound together prior topolymerization, can also be carried out according to known methods. Thebinding properties of the MIPs formed by either of the above methods canbe examined by re-binding of the template molecule.

The polymerization is performed in the presence of a pore-formingsolvent, a porogen. In order to stabilize the electrostatic interactionsbetween the functional monomers and the template compound the porogen isoften chosen from among aprotic solvents of low to moderate polarity.Template compounds often exhibit moderate to high solubility in thepolymerization media and these, or their structural analogues, cantherefore be utilized directly in this standard procedure.

While it is possible to use the targeted molecule itself as thetemplate, a structural analog of the target molecule is commonlypreferred because: (a) the targeted molecule may be unstable under thepolymerization conditions or may inhibit the polymerization; (b) thetargeted molecule may not be available in sufficient quantities due tocomplexity of its synthesis or cost, or both; (c) the template may beinsoluble or poorly soluble in the pre-polymerization mixture; (d) theMIP may remain contaminated by low levels of the targeted moleculeretained in poorly accessible regions of the polymer matrix, which maybleed from the MIP during use; and/or (e) the target analyte(s) maypresent a significant health risk and should not be used as atemplate(s). In the case of nitroso-compounds, particularly thecompounds known as TSNAs described below, it is often more convenient touse functional analogues thereof as template compounds. For example,sulfonamide, enamine, or amide, e.g. formamide, derivatives of TSNAs canbe template compounds, see FIG. 2 for examples of the same.

Where the MIP is derived using a functional analog of the targetedcompound, the functional analogue should be isosteric and preferablyalso isoelectronic with the targeted compound, or it may contain asubstructure of the targeted compound where strong interactions may belikely.

As used herein a “structural analogue” of a molecule is not identical tothe original molecule, but is in part or whole similar to part or all ofthe original molecule in terms of molecular shape, electron distributionor other characteristics.

Nitroso-containing compounds, particularly nitrosamines, which have thegeneral formula O═N—N(R₁)(R₂) are among the numerous ingredients oftobacco and tobacco smoke that have been suggested as having a harmfuleffect on consumers. Of interest for the present invention is the groupof nitrosamines that occur naturally in tobacco, TSNAs, see FIG. 2.

Possible isosteric analogs for the targeting of nitrosamines are seen inFIG. 3. The molecules shown are all derivatives of the parent amine andcan be synthesized in a single step from the secondary amine andcorresponding aldehyde or acid chloride. Molecular models of the enamine(FIG. 4B) have shown a good steric complementarity with NNAL.

The MIPs described in WO 05/112670 show promising results when used foranalysis and extraction of numerous nicotine metabolites from analyticalsolutions, body fluids, and tobacco materials. However, as TSNAs presenta specific and narrow field of interest, investigation as to new ways torecover these compounds from various materials is ongoing. In thatregard, MIP formation using new materials and methods has beenevaluated.

Design of new MIPs started with choice of a suitable template. As notedabove, the template imparts selectivity to the polymer and shouldideally be chemically stable, readily available, easy to handle, andimpart selective binding properties. As one goal of the invention is toreduce human exposure to nitrosamines they were not potential templates.Instead, formamides, enamines and sulfonamides (see FIG. 2) could beused to replace the nitroso group given their similar geometrics andpossession of a partial negative charge in the same position.

The monomer, cross-linker and polymerization conditions (e.g., solventor porogen, initiator, and temperature) also influence properties of thefinal MIP. Monomers evaluated were the acidic MAA as well as the neutralmonomer 2-hydroxyethylmethacrylate (HEMA), see FIG. 5. The cross-linker,which eventually makes up the bulk of the polymer, also influenceswhether the polymer is hydrophilic or hydrophobic. Thus the hydrophiliccross-linker PETRA and the hydrophobic EDMA were evaluated, see FIG. 6.For polymerization, both thermal and photochemical initiation wereevaluated. Initial experiments indicated that neutral, hydrophobic MIPsimprinted with an enamine or sulfonamide and prepared using UVpolymerization showed surprising results. Follow on analysis wasconducted which included an acidic and a hydrophilic MIP for comparison,results are summarized below.

By way of explanation and not of limitation, the invention will befurther described in more detail with reference to a number of examples.The invention refers to template molecules, polymer materials designedto bind TSNAs present in organic or aqueous systems, and finally use ofsaid materials in, for example, analytical or preparative separations,in chromatography, for analytical sample pre-treatment, and in chemicalsensors. Unless otherwise described, materials are commerciallyavailable or can be prepared by conventional techniques.

Example 1 Preparation of MIPs for Evaluation

Twelve different MIPs were prepared which represented all possiblecombinations of three templates (formamide, enamine, and sulfonamide),two monomers (acidic and neutral), and two cross-linkers (hydrophilicand hydrophobic). Using a 1:1 mixture of N-nitrosopiperidine and(−)-nicotine in water the MIPs were evaluated. Non-imprinted referencepolymers were also generated and evaluated under like conditions.Results are summarized in FIG. 7. Examples of the preparation of enamineand pyridine carbinol templates as well as further description ofmethods which can be used for the purposes of the present invention canbe found in WO 05/112670.

As is evident from FIG. 7, MIPs using acidic monomers bind large amountsof nicotine. For applications where TSNAs are to be screened out whilenicotine levels should remain unaffected, then, such a monomer is lessfavored. In addition, it is evident that MIPs with a hydrophobiccross-linker are better at binding nitrosamine than hydrophilic MIPs.

Example 2 Preparation of MIPs for Analytical Comparison

In view of the surprisingly positive performance of neutral, hydrophobicMIPs as summarized in FIG. 7, the neutral, hydrophobic MIPs imprintedwith enamine or sulfonamide templates and polymerized using UV wereselected for further analysis. For comparison, the best-performinghydrophilic MIP was also included in the sample. Seven MIPs wereprepared using the parameters summarized in Table 1.

TABLE 1 Name Template Monomer Cross-Linker MIP 1

HEMA EDMA MIP 2

PETRA PETRA MIP 3

HEMA EDMA MIP 4 NNAL analogue HEMA EDMA MIP 5 NNAL analogue HEMA EDMAMIP 6

HEMA EDMA MIP 7

HEMA EDMA

Example 3 Evaluation of Selected MIPs with TSNA/Nicotine Mixtures

SPE columns were prepared, each containing 25 mg of MIPs 1-7. To eachcolumn 1 ml of aqueous solution containing 0.30 μg/ml total TSNA and 0.2μg/ml nicotine was added. HPLC was used to determine the amount of TSNAthat had not been extracted in each column, allowing for calculation ofthe extracted material, see FIG. 8. 1 ml of water was then passedthrough each column and the amount of TSNA released, if any, wasdetermined see FIG. 9. Each of FIGS. 8 and 9 represent the average oftwo experiments.

Based on the strong performance of the hydrophobic, neutral MIPs, theevaluation for MIPs 1 and 3-7 was repeated using 1 ml of test solutioncontaining 0.30 μg/ml total TSNA and 4 μg/ml nicotine in pH 6.3phosphate buffer (ionic strength 0.09). Results are shown in FIGS. 10and 11, where both represent an average of two experiments. As can beseen, MIPs 1, 3, and 4 performed best by retaining 100% of the TSNAwhile allowing most of the nicotine to pass unencumbered.

The performance of MIPs 1, 3, and 4 was also evaluated at pH 5.3 and7.3. As pH increased, retention of nicotine increased from approximately10% to 30%, indicating that where nicotine is not to be affected,optimal performance is attained using slightly acidic pH. An acidic MIPformed using the acidic monomer MAA and the hydrophobic cross-linkerEDMA was also evaluated using the same conditions at pH 6.3, it retainedabout 90% of the nicotine.

Regeneration of the MIPs was performed using a 0.5% TFA in MeOH wash.Other acid/alcohol mixtures may be used as well.

Example 4 Evaluation of Select MIPs with Specific TSNAs and Nicotine

After finding the surprisingly positive performance of MIPs 1, 3, and 4in the above-described experiments, a further evaluation was conducted.SPE columns were prepared with 25 mg of ground MIP and five loads oftest solution at 1 ml each were loaded on the columns. The test solutionwas ca. 80 ng/ml each of NNN, NNK, NAT, 40 ng/ml NAB and 4 μg/mlnicotine in pH 6.3 phosphate buffer (ionic strength 0.09). After loadingeach sample the amount of unretained nicotine and each TSNA wasdetermined using HPLC. A control non-imprinted polymer was alsoevaluated. Results are shown in FIGS. 12-16. As seen in the figures,about 10% of the nicotine is bound by the MIPs in the first loadingstep. This saturates the MIP with nicotine and adding more sample on thecolumn does not result in a further significant retention of nicotine.NNN is most weakly bound to the MIP and breakthrough is seen in thesecond loading step. Each of MIPs 1, 3, and 4 is excellent at retainingNNK, NAB, and NAT. Thus, the combination of neutral functional monomer,enamine or sulfonamide template, and hydrophobic cross-linker results ina surprisingly high retention of TSNA from a mixed sample whileretaining only a minimal amount of nicotine.

Such MIPs are particularly attractive for applications where TSNAs areto be removed from a sample but nicotine should not be affected, such astreatment of tobacco or tobacco smoke to remove TSNAs. In addition, suchMIPs can be utilized in an analytical capacity to measure the amount ofTSNA is a sample of a product or a sample from a patient. In such cases,if nicotine is a component of interest the amount of nicotine retainedby the MIP can be quantified and other methods, for example,nicotine-specific MIPs can be used to quantify the remaining amount ofnicotine in the sample. The further step of evaluating nicotine levelscould be done prior or subsequent to use of the TSNA specific MIPs ofthe present invention.

Example 5 Use of a MIP of the Invention in the Treatment of TobaccoExtracts

The polymer produced according to the parameters described above can beincorporated into a SPE column and the column can be conditioned asnecessary. Neutral functional monomers used in the polymer could be, forexample, HEMA, acrylamide, methacrylamide, N-methacrylamide, glycerolmonoacrylate, glycerol monomethacrylate, or2-(4-Vinylphenyl)-1,3-propanediol. Hydrophobic cross-linkers could be,for example, EDMA, TRIM, DVB, m-diisopropenylbenzene, tetramethyleneglycol dimethacrylate, pentaerythrithol tetraacrylate,N,N′-Methylenebisacrylamide, N,N′-Ethylenebisacrylamide,N,N′-Buthylenebisacrylamide, N,N′-Hexamethylenebisacrylamide. Furtherapplicable materials are known, see, for example, Molecularly ImprintedMaterial: Science and Technology, Yan, M; Ramström, O; Eds., MarcelDekker, New York, 2005.

Cut or shredded tobacco leaf can be extracted with water for 15-25minutes at 60° C. The tobacco is separated from the solution byfiltration and dried. The solution is passed through the SPE column andTSNA is absorbed from the extract. The column is then drained and thesolution concentrated by film evaporation, the concentrate is thenrecombined with the extracted tobacco and dried in air. Performance ofthe MIP can be evaluated by eluting bound compounds from the MIP using2×1 ml methanol containing 0.5% TFA and extract analyzed using HPLC-UV.

Example 6 Use of a MIP of the Invention in the Treatment of TobaccoExtracts

Using a continuous extraction process, US Blend-type shredded tobaccoleaf is loaded into a first extraction chamber into which super-criticalcarbon dioxide is fed. After contacting the tobacco, the carbon dioxideis fed into a second extraction chamber containing a MIP according tothe invention. Having contacted the polymer, the carbon dioxide isreturned to the first extraction chamber and contacted again with thetobacco. The cyclic process is continued until the TSNA content of thetobacco has been reduced to a desired level, whereupon the carbondioxide is vented from the system, and the tobacco removed from thefirst chamber. The MIP in the second chamber is then regenerated forreuse.

Example 7 Use of an MIP of the Invention for Sample Analysis

A SPE column is prepared by adding 25 mg of MIP according to theinvention. A test sample is added to the column, for example 5 ml ofhuman urine potentially containing TSNAs. The sample is allowed to passthrough the column, which would then be subjected to vacuum to removeall liquid and ensure the MIP material is dry. A wash may be conductedto remove any interfering compounds that may have non-specificallyassociated with the MIP, for example 1 ml distilled water. After dryingthe TSNAs can be recovered from the MIP using, e.g., 1 ml DCM andquantified using HPLC.

Example 8 Use of an MIP of the Invention in Smoking Articles

Referring to the drawings, FIGS. 17 and 18 illustrate smoking articlesin the form of cigarettes having a rod 1 of tobacco encased in a wrapper2 attached to a smoke filter 3 by means of a tipping paper 4. Forclarity, the tipping paper 4 is shown spaced from the wrapper 2, but inpractice they lie in close contact.

In FIG. 17, the smoke filter 3 comprises three cylindrical filterelements 3 a, 3 b, 3 c. The first filter element 3 a at the mouth end ofthe filter is 7 mm in length, composed of cellulose acetate towimpregnated with 7% by weight of triacetin plasticizer having a 25 mmwater gauge pressure drop over its length. The second filter element 3b, positioned centrally is a cavity 5 mm in length containing 150 mg ofactivated carbon granules. The third filter element 3 c adjacent the rod1 is 15 mm in length, has a 90 mm water gauge pressure drop over itslength, and comprises 80 mg cellulose acetate tow. The tow isimpregnated with 4% by weight of triacetin and has 80 mg of MIP specificfor TSNAs as described herein, distributed evenly throughout its volumein a “Dalmatian” style.

The cigarette shown in FIG. 18 is similar to that of FIG. 17 except thatthe smoke filter 3 has four coaxial, cylindrical filter elements 3 a, 3b, 3 c and 3 d. The first filter element 3 a at the mouth end of thecigarette is 5 mm in length, and composed of cellulose acetate towimpregnated with 7% by weight of triacetin plasticizer. The secondfilter element 3 b, positioned adjacent the first filter element 3 a isa cavity 5 mm in length containing 200 mg of MIP specific for TSNAs,produced as described herein. The third filter element 3 c adjacent thesecond filter element 3 b is 10 mm in length and comprises celluloseacetate tow impregnated with 7% by weight of triacetin. The fourthfilter element 3 d lies between the third filter element 3 c, is 7 mm inlength and comprises 80 mg of granular activated carbon. A ring ofventilation holes 5 is formed in the tipping paper 4 in a radial planeA-A which deliver air into the third filter element 3 c about 3 mmdownstream of the junction with the fourth filter 5 element 3 d whensmoke is inhaled through the cigarette.

The foregoing disclosure has been set forth merely to illustrate theinvention and is not intended to be limiting. Since modifications of thedisclosed embodiments incorporating the spirit and substance of theinvention may occur to persons skilled in the art, the invention shouldbe construed to include everything within the scope of the appendedclaims and equivalents thereof.

The invention claimed is:
 1. A molecularly imprinted polymer selectivefor at least one tobacco specific nitrosamine (TSNA), the polymer havingbeen prepared using materials comprising: a TSNA or a structuralanalogue thereof; a neutral functional monomer; and a hydrophobiccross-linker.
 2. A polymer according to claim 1, wherein said structuralanalogue of a TSNA is an enamine analogue of a TSNA.
 3. A polymeraccording to claim 1, wherein said structural analogue of a TSNA is anamide analogue of a TSNA.
 4. A polymer according to claim 1, whereinsaid structural analogue of a TSNA is a sulfonamide analogue of a TSNA.5. A polymer according to claim 1, wherein the polymer is selective forNNK.
 6. A polymer according to claim 1, wherein the polymer is selectivefor NNA.
 7. A polymer according to claim 1, wherein the polymer isselective for NNN.
 8. A polymer according to claim 1, wherein thepolymer is selective for NAB.
 9. A polymer according to claim 1, whereinthe polymer is selective for NAT.
 10. A polymer according to claim 1,wherein the polymer is selective for NNAL.
 11. A polymer according toclaim 1, wherein the polymer is selective for iso-NNAL.
 12. A polymeraccording to claim 1, wherein the polymer is selective for iso-NNAC. 13.A kit, comprising: a molecularly imprinted polymer selective for atleast one tobacco specific nitrosamine (TSNA), the polymer preparedusing materials comprising: a TSNA or a structural analogue thereof; aneutral functional monomer; and a hydrophobic cross-linker; andinstructions for using the molecularly imprinted polymer to perform atleast one of detecting, quantifying, and separating nitrosamines in asample.
 14. A method of preparing a molecularly imprinted polymerselective for TSNAs, comprising: co-polymerizing at least one neutralfunctional monomer and at least one hydrophobic cross-linker in thepresence of at least one TSNA structural analogue in a polymerizationmedium containing at least one free radical initiator to produce amolecularly imprinted polymer bound to a TSNA structural analogue; andremoving the TSNA structural analogue from the molecularly imprintedpolymer.
 15. A molecularly imprinted polymer produced by the method ofclaim 14.